High sensitivity, real-time, label-free monitoring of binding to a surface in a liquid, in a gaseous environment, or in a vacuum has a wide range of applications, including thin film thickness monitoring during deposition, biological applications for molecular, viral, bacterial and cellular detection, surface science, surfactant research, drug research and discovery, electrochemistry (including plating and etching) and in situ monitoring (eg. oil condition).
Diagnostic tests based on a binding event between members of binding pair are widely used in medicine, agriculture, food analysis and research. These tests are designed to detect the presence, amount, rate of binding of a wide variety analytes. Typical binding pairs include antibody-antigen, receptor-ligand, DNA or RNA hybridizing pairs.
In a solid-phase assay molecules are immobilized on a solid surface, the solid surface is exposed to a sample under conditions that promote binding, binding occurs in a defined detection zone, and binding events may be detected by a variety of direct and indirect methods. Methods of direct detection include a change in mass, viscosity, elasticity, dissipation, electric charge or potential, surface stress, reflectivity, thickness, color. Methods of indirect detection include the use of a chromophore or fluorescent label, and radiolabels. Further, binding can be detected after it occurs by a secondary fluorescent-labeled anti-analyte antibody.
Prior art U.S. Pat. No. 5,804,453, “Fiber optic direct-sensing bioprobe using a phase-tracking approach” issued to Chen; Duan-Jun, and related U.S. Application No. 20050254062 “Fiber-optic assay apparatus based on phase-shift interferometry” issued to H. Tan et al. discloses an optical fiber interferometer assay device that is designed for direct detection of binding to an optical fiber end surface. Detection is based on a change in thickness at the optical fiber end surface due to molecular binding. The change in thickness changes an optical interference signal due to the phase shift between light reflected from two layers on the optical fiber end surface: a first layer and a second layer that is directly exposed to the sample. The prior art optical fiber interferometer analyzes the phase shift between the first and second layers using an optical spectrometer operating with visible light in the range 450-700 nm. The phase shift due to binding is detected by changes in the reflected light spectrum over a range of wavelengths, specifically, changes in the peaks and valleys of the reflected light optical spectrum.
One limitation of the prior art optical fiber interferometer is the relatively low sensitivity. The measured phase shift depends on a change in refractive index and the change in refractive index due to binding of the analyte molecules is extremely small. As a result a large number of molecules are needed to produce a detectable signal. In addition, small changes in the positions of peaks and valleys in the optical spectrum are difficult to detect, and the signal may be weak and buried in noise.
Prior art U.S. Pat. No. 6,436,647, “Method for detecting chemical interactions between naturally occurring biological analyte molecules that are non identical binding partners” issued to Quate, C. F. et al. discloses a method of using cantilevers as sensors for detecting chemical interactions between naturally occurring bio-polymers which are non-identical binding partners. The method is useful whether the reactions occur through electrostatic forces or other forces. Induced stress, heat, or change in mass is detected where a binding partner is placed on a cantilever for possible reaction with analyte molecules (i.e., a non-identical binding partner). The method is particularly useful in determining DNA hybridization but may be useful in detecting interaction in any chemical assay.
Also, prior art U.S. Pat. No. 6,289,717, “Micromechanical antibody sensor”, issued to Thundat et al. discloses a sensor apparatus using a microcantilevered spring element having a coating of a detector molecule such as an antibody or antigen. A sample containing a target molecule or substrate is provided to the coating. The spring element bends in response to the stress induced by the binding which occurs between the detector and target molecules. Deflections of the cantilever are detected by a variety of detection techniques. The microcantilever may be approximately 1 to 200 .mu.m long, approximately 1 to 50 .mu.m wide, and approximately 0.3 to 3.0 .mu.m thick. Sensitivity for detection of deflections is in the range of 0.01 nanometers.
One disadvantage of cantilever deflection based sensors is relatively low sensitivity due to the fact that a detectable cantilever deflection is caused by the cumulative effect of the binding of a large number of molecules to a cantilever surface. This can take a significant amount of time, and therefore the sensor response may be relatively slow. Further, the slow response leaves the sensor susceptible to a variety of noise sources (drift, thermal noise). In addition, the cantilever is a relatively rigid free-standing element, rather like a swimming pool diving board and as a result, the surface mass density of typical cantilever sensors is much greater than the surface mass density of the molecules bound to its surface.
In addition, the cantilever sensor has an inconvenient form factor. Cantilever sensors may require an optical alignment each time they are replaced. This is generally inconvenient and particularly inconvenient in the case of multiple cantilever sensors forming an array. Further, cantilever sensors may be too expensive for operation as a single-use disposable, and re-use over many cycles may be difficult to achieve.
U.S. Pat. No. 5,807,758, “Chemical and biological sensor using an ultra-sensitive force transducer”, issued to Lee et al. discloses a method and apparatus for detecting a target species. The target molecule may be in liquid phase (in solution) or (for some embodiments of the invention) in vapor phase. A sensor according to the present invention monitors whether a target species has selectively bound to groups on the cantilever surface by monitoring the displacement of the cantilever, and hence the force acting on the cantilever. This force acting on the cantilever arises from the force acting on a structure that moves in electric or magnetic field, and that may be selectively bound to the cantilever. In the case of target species having a sufficiently large net electric charge or dipole moment, the target species itself may serve as the structure that moves in an electric field. More typically however, separate modified structures, such as modified magnetic beads or modified beads having a net charge or a dipole moment, will, when selectively bound to the cantilever, exert a force on the cantilever that relates to the presence of the target species.
This is a cantilever-based approach that has the additional disadvantage of requiring a label and thus it provides only an indirect measure of analyte binding.
U.S. Application No. 20040096357, “Composite sensor membrane”, issued to Majumdar et al. discloses a sensor including a membrane to deflect in response to a change in surface stress, where a layer on the membrane is to couple one or more probe molecules with the membrane. The membrane may deflect when a target molecule reacts with one or more probe molecules.
This is a membrane sensor that operates on the same principle the cantilever sensors to detect analyte binding by changes in surface stress. It has the disadvantages of the cantilever sensors as previously described. Specifically, the sensitivity may be relatively low because it requires a large number of binding events. The sensor response may be relatively slow and is generally susceptible to noise and drift. In addition, the membrane is a relatively rigid free-standing element and the surface mass density of the membrane may be much greater than the surface mass density of the molecules bound to its surface. Further, the membrane sensor may be too expensive to be a single-use disposable, and re-use over many cycles may be difficult to achieve.
A related sensor, the quartz crystal microbalance (QCM) is an electro acoustic method suitable for mass and viscoelastic analysis of adsorbed protein layers at the solid/water interface. A typical QCM sensor consists of a megahertz piezoelectric quartz crystal sandwiched between two gold electrodes. The crystal can be brought to resonant oscillation, and shear motions by means of A/C current between the electrodes. Since the resonant frequency (f) can be determined with very high precision, usually less than 1 Hz, the adsorbed mass at the QCM-surface can be detected, or “balanced”, down to a few ng/cm2. It has also been shown that there is linear relation between the adsorbed rigid mass and the change in f, in an ideal air/solid situation.
U.S. Pat. No. 6,006,589 “Piezoelectric crystal microbalance device”, issued to Rodahl et al. discloses a device and a process for measuring resonant frequency and dissipation factor of a piezoelectric resonator. After exciting the resonator to oscillation, the driving power to the oscillator is turned off after the decay of the oscillation of the resonator is recorded and used to give a measure of at least one of the resonators properties, such as dissipation factor, changes in the dissipation factor, resonant frequency and changes in the resonant frequency. The invention allows these measurements to be performed at either the fundamental resonant frequency or one (or more) of the overtones. The device and the process disclosed herein may be used in a variety of applications such as, for example, measurement of phase transitions in thin films, the detection of adsorption of biomolecules, and measurements of the viscoelastic properties of thin films.
The crystal microbalance sensor typically vibrates in a shear mode with an amplitude of about 1 nm at a fundamental resonant frequency given by:fres˜1/dwhere d is the thickness of the quartz plate. For example, if d is 0.17 mm, the resonance frequency is approximately 10 MHz. The quartz sensor starts to oscillate if an AC electric field with a frequency centered close to the fundamental resonant frequency of the sensor is applied perpendicularly to its surfaces. Usually, electrodes on each side of the sensor plate are deposited by evaporation and are subsequently contacted to an external AC field generator (for example to a signal generator, or to an oscillator drive circuit, or the like). Under favorable conditions this arrangement is capable of sensing mass changes smaller than 1 ng/cm^2.
Ideally the mass changes at the sensor electrode(s) induce a shift in the resonance frequency of the sensor, proportional to the mass changes:Delta M=−C Delta fres where C, the proportionality constant, depends on the thickness of the quartz plate.
This relation is valid provided that the mass is attached rigidly to the electrode and follows the oscillatory motion of the sensor without dissipative losses. The relation may fail when the added mass is viscous or is not rigidly attached to the electrode(s) and can thus suffer elastic or plastic deformation(s) during oscillations. The relation between added mass and the shift of the resonant frequency then becomes more complex. The latter situation arises when for example a water droplet is deposited onto an electrode of the quartz sensor.
The Sauerbrey equation describes the linear relation between frequency changes and changes in mass for thin films adsorbing to the crystal microbalance sensor surface. It gives a good estimation of film thickness, as long as the dissipation is relatively low. When the dissipation value reaches above 1×10−6 per 5 Hz, the film is too soft to function as a fully coupled oscillator. A calculated thickness value will hence be somewhat less than the true value.
Proteins at the water/QCM surface interface can also be quantified with resonance frequency determination, but adsorbed protein layers also have some degree of structural flexibility or viscoelasticity, that are invisible to a simple resonance frequency determination. Viscoelasticity can, however, be visualized by measuring the energy loss, or dissipation (D) of the shear movement of the crystal in water. A new principle of measuring D is to drive the crystal with A/C current at the resonant f followed by disconnection and analysis of the resulting damped sinusoidal curve. This development of pulse assisted discrimination of resonance frequency and dissipation makes QCM analysis of adsorbed protein layers very simple and gives unique information about the hydrodynamic conductivity of the adsorbed protein layers and surrounding water. Very small structural and orientation changes of an adsorbed protein layer, including chemical cross-linking, may be monitored with high accuracy.
One disadvantage of this approach is the piezoelectric crystal sensor size—typically 10-30 mm diameter and 0.1-0.5 mm thickness. Also, the entire sensor element including piezoelectric crystal and electrodes is typically thrown out after each use. In some cases, the sensor element can be reused a number of times, but this requires careful cleaning. In addition, sensor size limits scalability to an array-sensing format for multiple analytes.
A preferred embodiment of the sensor apparatus of the invention includes an optical-fiber based interferometer to measure small displacements. One such interferometer is disclosed in U.S. Pat. No. 5,017,010, “High sensitivity position sensor and method”, issued to Mamin et al. This is a highly sensitive apparatus for sensing the position of a movable member comprises an optical directional coupler providing four external ports.
Light from a short coherence length diode laser is injected into the first port. The coupler serves as a beam splitter to direct one portion of the injected light to the member via the second port and a single mode optical fiber. Part of this one portion is reflected concurrently from the member and from the adjacent polished coating at the end face of said fiber back into said fiber and optically coupled via the third port to a photodetector to provide a signal whose amplitude is indicative of the position of the member, based upon the relative phase of said concurrent reflections. The other portion of the injected light is optically coupled to and via the fourth port to another photodetector for providing, as a reference, a signal proportional to the intensity of the injected light. These two signals are supplied to a subtractive circuit for providing an output in which power fluctuations of the laser are minimized.
Another embodiment of the invention uses a laser-diode based interferometer for direct sensing of small displacements. One such laser-diode based interferometer is disclosed in U.S. Pat. No. 5,189,906, “Compact atomic force microscope”, issued to Sarid et al. This is an atomic force microscope using a laser diode and optical interference of light reflected back into the laser to measure the vertical position of a sensing tip wherein the sensing tip can be either off the sample surface and vibrated where changes in the amplitude of vibration near the natural frequency of the cantilever are used as a measure of changes of electric or magnetic force on the sensing tip; or, the sensing tip can be placed on the sample surface with no vibration to measure directly the profile of the sample surface.